Platelet aggregation plays a fundamental role in the hemostatic response, and in thrombotic disease (Colman, R. W. and Walsh, P. N. (1987) in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, Colman, R. W., Hirsch, J., Marder, V. J., and Salzman, E. W., eds. (J. B. Lippincott Company, Philadelphia), pp. 594-605; and Stein, B., Fuster, V., Israel, D. H., Cohen, M., Badimon, L., Badimon, J. J., and Chesebro, J. H. (1989) J. Am. Coll. Cardiol. 14, 813-836). It is believed that the first step in platelet aggregation involves activation of platelets, however, the mechanisms of platelet activation and subsequent aggregation are complex, characterized by numerous intracellular pathways and extracellular ligands and binding sites. It is known, however, that glycoprotein II.sub.b III.sub.a (GP II.sub.b III.sub.a ), a Ca.sup.+2 dependent heterodimeric receptor (Phillips, D. R., Charo, I. F., Parise, L. V., and Fitzgerald, L. A. (1988) Blood, 71, 831-843; and Plow, E. F. and Ginsburg, M. H. (1989) in Progress in Hemostasis and Thrombosis, Coller, B. S., ed., (W. B. Saunders Company, Philadelphia), vol 9, pp. 117-156), when exposed on the surface membrane of activated platelets, can bind different adhesive proteins such as fibrinogen (Fg), fibronectin, von Willebrand factor, or vitronectin. The binding of fibrinogen to platelets via GP II.sub.b III.sub.a mediates aggregation and is critical to the formation of a hemostatic plug at an injured vessel wall. This interaction is considered to be the final common step of all platelet aggregation pathways and therefore provides an ideal target for therapeutic intervention in thrombotic disorders. The specific binding mechanism for the interaction of GP II.sub.b III.sub.a to its ligands is unknown; however, it is thought to be manifested through an Arg-Gly-Asp (RGD) sequence, an adhesion site recognition sequence (Ruoslahti, E. and Pierschbacher, M. D. (1987), Science 238, 491-497) common to the adhesive proteins that bind to GP II.sub.b III.sub.a.
Fibrinogen contains two RGD sequences at A.alpha.95-97 and A.alpha.572-574 (Doolittle, R. F., Watt, K. W. K., Colltrell, B. A., Strong, D. D. and Riley, M. (1979) Nature 280, 464-468). A third region of fibrinogen, corresponding to residues 400-411 of the gamma chain carboxy terminus, has also been implicated in binding to GP II.sub.b III.sub.a (Kloczewiak, M., Timmons, S., Lukas, T. J., and Hawiger, J. (1984) Biochemistry 23, 1767-1774). Evidence for the involvement of both the RGD and gamma chain regions in binding with GP II.sub.b III.sub.a is largely derived from binding and inhibition data and from studies with synthetic RGD peptides (Gartner, T. K. and Bennett, J. S. (1985) J. Biol. Chem. 260, 11891-11894; Plow, E. F., Pierschbacher, M. D., Ruoslahti, E., Marguerie, G. A., and Ginsberg, M. H. (1985) Proc. Nat. Acad. Sci. USA 82, 8057-8061; Haverstick, D. M., Cowan, J. F., Yamada, K. M., and Santoro, S. (1985) Blood 66, 946-952; and D'Souza, S. E., Ginsberg, M. H., Lam, S. -C. T., and Plow, E. F. (1988) J. Biol. Chem. 263, 3943-3951) and gamma chain peptides (Kloczewiak, M., Timmons, S., Bednarek, M. A., Sakon, M., and Hawiger, J. (1989) Biochemistry 28, 2915-2919).
It is known that proteins containing the peptide sequence RGD may be recognized as binding ligands of a number of cell adhesion receptors other than GP II.sub.b III.sub.a. These cell adhesion receptors comprise a family of heterodimeric protein receptors known as the integrins (Ginsberg, M. H., Loftus, J. C., and Plow, E. F. (1988) Thrombosis and Haemostasis 59, 1-6; and Hynes, R. O. (1988) Cell 48, 549-554). Among these receptors shown to bind RGD containing ligands are the vitronectin receptors (VnR) and the fibronectin receptors (FnR) (Pytela et al. (1985) Proc. Natl. Acad. Sci., USA 82, 5766-5770; Pytela et al. (1985) Cell 40, 191-198; and Sanchez-Madrid et al. (1983) J. Exp. Med. 158, 1785-1803). Furthermore, it is believed that other integrin receptors may be discovered which interact with RGD containing proteins. Thus, RGD containing therapeutics may antagonize a number of protein ligand-receptor interactions.
Many proteins affecting hemostasis have been purified and characterized from natural sources including both snake venoms and leeches. In view of the complexity of platelet aggregation, it is believed that the agents found in these natural sources mediate their hemostatic effects through a variety of mechanisms, which include fibrinolysis (Stocker, K. F. (1990) in Medical Uses of Snake Venoms, Stocker, K. F. ed., (CRC Press, Boca Raton), pp. 97-160; and Malinconico, S. M., Katz, J. B., and Budzynski, A. (1984) J. Lab. Clin. Med. 103, 44-58), the inhibition of thrombin (Markwardt F. (1988) in Hemostasis and Animal Venoms, H. Pirkle and F. G. Markland, Jr.,eds., (Marcel Dekker, New York), pp. 225-269) and factor Xa (Nutt, E., Gasic, T., Rodkey, J., Gasic, G. J., Jacobs, J. W. and Friedman, P. A. (1988) J. Boil. Chem. 263, 10162-10167; and Condra, C., Nutt, E., Petroski C. J., Simpson, E., Friedman, P. A., and Jacobs, J. W. (1989) Thromb. Haemostas. 61, 437-441), as well as antagonism of GP II.sub.b III.sub.a receptor binding to Fg (Huang, T. F., Holt, J. C., Kirby, E. P., and Niewiarowski, S. (1989) Biochemistry 28, 661-666; Gan, Z. R., Gould, R. J., Jacobs, J. W., Friedman, P. A., and Polokoff, M. A. (1988) J. Biol. Chem. 263, 19827-19832; Chao, B. H., Jakubowski, J. A., Savage, B., Chow, E. P., Marzec, U. L., Harker, L. A., and Maraganore, J. M. (1989) Proc. Natl. Acad. Sci. USA 86, 8050-8054; Shebuski, R. J. Ramjit, D. R. Bencen, G. H., and Polokoff, M. A. (1989) J. Biol. Chem. 264, 21550-21556; Dennis, M. S., Henzel, W. J., Pitti, R. M., Lipari, M. T., Napier, M. A., Deisher, T. A., Bunting, S. and Lazarus, R. A. (1990) Proc. Natl. Acad. Sci. USA 87, 2471-2475; Williams, J., Rucinski, B., Holt, J., and Niewiarowski, S. (1990) Biochim. Biophys. Acta 1039, 81-89; Musial, J., Niewiarowski, S., Rucinski, B., Stewart, G. J., Cook, J. J., Williams, J. A., and Edmunds Jr., L. H. (1990) Circulation 82, 261-273; and Seymour, J. L., Henzel, W. J., Nevins, B., Stults, J. T., and Lazarus, R. A. (1990) J. Biol. Chem. 265, 10143-10147).
Recent studies have established that the GP II.sub.b III.sub.a receptor antagonists from the venoms of pit vipers, which all contain the RGD sequence, constitute a general class of homologous proteins that are potent inhibitors of platelet aggregation (Dennis, M. S., Henzel, W. J., Pitti, R. M., Lipari, M. T., Napier, M. A., Deisher, T. A., Bunting, S. and Lazarus, R. A. (1990) Proc. Natl. Acad. Sci. USA 87, 2471-2475; co-pending application U.S. Ser. No. 07/362,718, filed Jun. 7, 1989; Huang, T. F., Holt, J. C., Kirby, E. P., and Niewiarowski, S. (1989) Biochemistry 28, 661-666; Gan, Z. R., Gould, R. J., Jacobs, J. W., Friedman, P. A., and Polokoff, M. A. (1988) J. Biol. Chem. 263, 19827-19832; and Chao, B. H., Jakubowski, J. A., Savage, B., Chow, E. P., Marzec, U. L., Harker, L. A., and Maraganore, J. M. (1989 Proc. Natl. Acad. Sci., USA 86, 8050-8054). These venom proteins are highly homologous to one another and constitute a family of related proteins that interact directly with GP II.sub.b III.sub.a, thereby blocking Fg binding (co-pending application U.S. Ser. No. 07/362,718, filed Jun. 7, 1989).
Leeches have long been known to possess agents that affect hemostasis (Sawyer, R. T. (1986) Leech Biology and Behaviour, Clarendon Press, Oxford, 2, 467-523; Sawyer, R. T. (1988) Hemostasis and Animal Vemons, H. Pirkle and F. G. Markland, Jr., eds., Marcel Dekker, New York, pp. 271-279) and several proteins have been isolated from leeches that affect hemostasis by various mechanisms. These include hirudin, a thrombin inhibitor from Hirudo medicinalis (Markwardt, F., (1988) Hemostasis and Animal Venoms, H. Pirkle and F. G. Markland, Jr., eds., Marcel Dekker, New York, pp. 255-269; Dodt, J., Seemuller, U., maschler, R., and Fritz, H. (1985) Biol. Chem. Hoppe-Seyler 366, 379-385); antistasin, a factor Xa inhibitor from Haementaria officinalis (Nutt, E., Gasic, T., Todkey, J., Gasic, G. J., Jacobs, J. W. and Friedman, P. A. (1988) J. Biol. Chem. 263, 10162-10167); as well as a similar factor Xa inhibitor from Haementaria ghilianii (Condra, C., Nutt, E., Petroski, C. J., Simpson, E., Friedman, P. A., and Jacobs, J. W. (1989) Thromb. Haemostas. 61, 437-441); and hementin, a fibrinolytic enzyme from Haementaria ghilianii (Malinconico, S. M., Katz, J. B., and Budzynski, A. (1984) J. Lab. Clin. Med. 103, 44-58).
More recently, it has been reported that leeches, and in particular the saliva of the medicinal leech, Hirudo medicinalis, have active factors that are able to inhibit platelet aggregation (Rigbi M., Levy, H., Eldor, A., Iraqu, F., Teitelbaum, Orevi, M., Horovitz, A., and Galun, R. (1987) Comp. Biochem. Physiol. 88C, 95-98); other leeches were also found to possess platelet aggregation inhibitors. In one instance, the platelet aggregation inhibitor was found to be a collagenase of molecular weight 50,000 (Sawyer, R. T., et al., International Patent Application No. PCT/GB86/00481). This collagenase has also been identified in a wide variety of leeches.
Although inhibitors of platelet aggregation isolated from leeches have been reported previously (e.g., Sawyer, R. T., et al., International Patent Application No. No. PCT/GB 86/00481; Rigbi M., Levy, H., Eldor, A., Iraqu, F., Teitelbaum, Orevi, M., Horovitz, A., and Galun, R. (1987) Comp. Biochem. Physiol. 88C, 95-98), these inhibitors act at other, or unknown, points in the aggregation pathways. Prior to the present invention, there are no known reports of inhibitors of platelet aggregation which acit via antagonism of the GP II.sub.b III.sub.a receptor that have been isolated from leeches. Thus, the present discovery represents the first description of GP II.sub.b III.sub.a antagonists isolated from leeches that act as platelet aggregation inhibitors.
The recognition, isolation, and characterization of anticoagulant factors in leeches is complicated by the difficulties of obtaining sufficient raw material for study. These difficulties are compounded by the extreme complexity of the clotting system. While it is not necessary for purposes of the present application to detail the multiple biochemical pathways that constitute the clotting process, a large number of extrinsic and intrinsic factors, some contributing to more than one pathway, are involved and are not fully understood. The multiplicity of elements contributing to clotting, and the consequent number of possible interactions and feedback loops, makes it extremely difficult to accurately assess the effects and mechanism of an exogenously added agent. Potential targets for inhibition of platelet aggregation, include; phospholipase A.sub.2, fibrinogen, thrombin, adenylate cyclase, cyclooxygenase, thromboxane synthase or receptor, and GP II.sub.b III.sub.a. There has been considerable recent interest in the GP II.sub.b III.sub.a receptor, the most thoroughly studied member of the integrin family of cell adhesion receptors. Since many thrombotic diseases may be mediated by platelet adhesion and aggregation, Inhibitors of the Fg/GP II.sub.b III.sub.a interaction have great potential as agents for therapeutic intervention in thrombotic disease (Stein, B., et al., (1989) J. Am. Coll. Cardiol. 14, 813-836).
Therefore, it is an object of the present invention to ascertain whether hematophagous leeches contain antithrombotic agents that act via inhibition of GP II.sub.b III.sub.a /Fg binding to inhibit platelet aggregation. To achieve the foregoing object, it is a further object to develop a specific assay to measure the inhibition of GP II.sub.b III.sub.a /Fg binding, as well as an assay to detect inhibition of in vitro platelet aggregation.
It is still a further object to isolate and purify novel antithrombotic agents from leeches, especially those that inhibit Fg binding to GP II.sub.b III.sub.a. Additionally, it is an object to provide synthetic methods for producing leech derived antithrombotics for therapeutic intervention. These and other objects will be apparent from consideration of this application as a whole.